Project Summary Long noncoding (lnc) RNAs are a new frontier that we must explore to fully understand human development. To date, the functions of only a small number of lncRNAs have been described in human development, and the continued existence of this fundamental gap in our knowledge of lncRNA function will impede the ability to control differentiation for cell-based therapies and understand how mutations in noncoding regions of the genome lead to disease. The long-term goal of this work is to elucidate the mechanisms by which lncRNAs regulate development of human definitive endoderm (DE) and use this insight to direct more efficient differentiation of gastrointestinal tissues for regenerative medicine. We have identified the lncRNA DIGIT and show that it regulates DE differentiation of human and mouse embryonic stem cells (ESCs). The gene encoding DIGIT is divergent to the gene encoding Goosecoid (GSC) in both humans and mice. In human ESCs, we find that DIGIT controls DE differentiation through regulation of GSC. The overall objective is to determine how DIGIT regulates development. The central hypothesis is that DIGIT regulates DE differentiation through different pathways in ESC differentiation and in vivo development. The rationale for this proposal is that understanding how DIGIT controls DE differentiation will make it possible to modulate expression of this lncRNA to facilitate differentiation of tissues for regenerative medicine and provide insight into the mechanisms by which lncRNAs regulate divergent genes. This proposal will also determine how the mouse ortholog of DIGIT (Digit) regulates differentiation and whether mouse models can teach us about the function of DIGIT when hESCs are differentiated for therapy. The central hypothesis will be tested by pursuing three specific aims: (1) determine how DIGIT regulates GSC expression in hESCs, (2) determine how DIGIT and protein partners interact to regulate DE differentiation, and (3) determine the role of Digit in mouse development. In the first aim, gain and loss of function analysis coupled with RNA fluorescent in situ hybridization and chromatin precipitation will determine how DIGIT controls GSC expression. In the second aim, we will investigate how protein partners interact with DIGIT to regulate differentiation. In the third aim, we will use mouse ESC culture and in vivo development to define the role of Digit in DE differentiation. The proposed work is significant because understanding the mechanism by which lncRNAs regulate DE differentiation will lead to transform- ative strategies for the differentiation of pancreatic, liver, intestinal and lung cells for therapeutics. It will also teach us how lncRNAs regulate divergently transcribed genes and whether mouse models for this lncRNA can be used to understand hESC differentiation. This work is innovative in the focus on lncRNAs to understand mechanism of development and in how it uses genome editing approaches to disrupt and track lncRNA transcription.